Impairments of cerebellar structure and function in a zebrafish KO of neuropsychiatric risk gene znf536.

Genetic variants in ZNF536 contribute to the risk for neuropsychiatric disorders such as schizophrenia, autism, and others. The role of this putative transcriptional repressor in brain development and function is, however, largely unknown. We generated znf536 knockout (KO) zebrafish and studied their behavior, brain anatomy, and brain function.

Behavioral analysis through the lifespan of mutant zebrafish identifies defects in sensorimotor transformation.

is a genetic risk factor for multiple psychiatric disorders. Compared to the dozens of murine models, there is a paucity of zebrafish models-an organism amenable to high-throughput experimentation. We conducted the longitudinal neurobehavioral analysis of mutant zebrafish across key stages of life.

Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition.

CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets.

A single-cell transcriptome atlas of the maturing zebrafish telencephalon.

The zebrafish telencephalon is composed of highly specialized subregions that regulate complex behaviors such as learning, memory, and social interactions. The transcriptional signatures of the neuronal cell types in the telencephalon and the timeline of their emergence from larva to adult remain largely undescribed. Using an integrated analysis of single-cell transcriptomes of approximately 64,000 cells obtained from 6-day-postfertilization (dpf), 15-dpf, and adult telencephalon, we delineated nine main neuronal cell types in the pallium and eight in the subpallium and nominated novel marker genes.

ZebraShare: a new venue for rapid dissemination of zebrafish mutant data.

Background: In the past decade, the zebrafish community has widely embraced targeted mutagenesis technologies, resulting in an abundance of mutant lines. While many lines have proven to be useful for investigating gene function, many have also shown no apparent phenotype, or phenotypes not of interest to the originating lab. In order for labs to document and share information about these lines, we have created ZebraShare as a new resource offered within ZFIN.

A Customizable Low-Cost System for Massively Parallel Zebrafish Behavioral Phenotyping.

High-throughput behavioral phenotyping is critical to genetic or chemical screening approaches. Zebrafish larvae are amenable to high-throughput behavioral screening because of their rapid development, small size, and conserved vertebrate brain architecture. Existing commercial behavioral phenotyping systems are expensive and not easily modified for new assays.

Macromolecular modeling and design in Rosetta: recent methods and frameworks.

The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities.

Phenotypic Landscape of Schizophrenia-Associated Genes Defines Candidates and Their Shared Functions.

Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologs of 132 human schizophrenia-associated genes. We created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities.

Kctd13 deletion reduces synaptic transmission via increased RhoA.

Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders and are among the most prevalent in autism spectrum disorders. Of many 16p11.

Internal guide RNA interactions interfere with Cas9-mediated cleavage.

The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo.

CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering.

In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing.

Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development.

Error-prone repair of DNA double-strand breaks (DSBs) has been postulated to occur through classical non-homologous end joining (NHEJ) in systems ranging from nematode somatic tissues to zebrafish embryos. Contrary to this model, we show that zebrafish embryos mutant for DNA polymerase theta (Polq), a critical component of alternative end joining (alt-EJ), cannot repair DSBs induced by CRISPR/Cas9 or ionizing radiation. In the absence of DSBs, polq mutants are phenotypically normal, but they do not survive mutagenesis and display dramatic differences in the mutation profiles compared with the wild-type.

Massively parallel determination and modeling of endonuclease substrate specificity.

We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models.

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method.

Reprogramming homing endonuclease specificity through computational design and directed evolution.

Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency.

Improved modeling of side-chain--base interactions and plasticity in protein--DNA interface design.

Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed "motifs") was described.

Mining endonuclease cleavage determinants in genomic sequence data.

Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold.

A synthetic homing endonuclease-based gene drive system in the human malaria mosquito.

Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito's ability to serve as a disease vector. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations.

Exploitation of binding energy for catalysis and design.

Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme-DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side.